Integrating Natural Deep Eutectic Solvents into Nanostructured Lipid Carriers: An Industrial Look

Abstract

The industries are searching for greener alternatives for their productions due to the rising concern about the environment and creation of waste and by-products without industrial utility for that specific line of products. This investigation describes the development of two stable nanostructured lipid carriers (NLCs): one is the formulation of a standard NLC, and the other one is the same NLC formulation associated with a natural deep eutectic solvent (NaDES). The research presents the formulation paths of the NLCs through completeness, which encompass dynamic light scattering (DLS), zeta potential tests, and pH. Transmission electron microscopy (TEM) and confocal microscopy were performed to clarify the morphology. Cytotoxicity tests with zebrafish were realized, and the results are complementary to the in vitro outcomes reached with fibroblast L132 tests by the MTT technique and the zymography test. Infrared spectroscopy and X-ray diffractometry tests elucidated the link between the physicochemical characteristics of the formulation and its behavior and properties. Different cooling techniques were explored to prove the tailorable properties of the NLCs for any industrial applications. In conclusion, the compiled results show the successful formulation of new nanocarriers based on a sustainable, eco-friendly, and highly tailorable technology, which presents low cytotoxic potential.

Keywords: NLC; NaDES; green nanotechnology.

Figure A1

DH and PDI results obtained using the DLS technique in ZetaSizer were evaluated in order to study the behavior of the formulation of different oils and butters. This can be verified in (A,B), respectively.

Figure A2

In (A), the test with different Brij^® O10 proportions in the SLN can be verified. In (B), the results obtained with different formulation proportions of butter and oil are shown. With the addition of oil to the formulation, the usage of Brij^® O10 could be reduced by 50%, as can be verified in (C).

Figure 1

(A) shows the temperature test performed with the SLN with just tucumã butter. In (B), the addition of the oil to the formulation is demonstrated, creating a reproductible NLC. In (C,E) the different test concentrations with NaDES A can be observed, which can be also verified for NaDES B in (D). In (F), the reproducibility of the nanocarrier with the addition of NaDES is verified. (G) exposes the different conductivity potential between both the nanocarriers with and without NaDES.

Figure 2

In (A,B), the results from the stability test for Jama at room temperature (25 °C) and refrigerator temperature (4 °C), respectively, are shown. (C,D) shows the results from the stability test for JamaNaDES also at room temperature (25 °C) and refrigerator temperature (4 °C), respectively.

Figure 3

NLC ultrastructures and size distribution. Transmission electron micrographs of Jama are shown in (A) and of JamaNaDES in (B). In (C), the size distribution frequency of the particles in the images is shown. The nanostructures observed were measured, and the frequency distribution of observed diameters of JamaNaDES are presented in (C), with the red line representing the log-normal distribution. The TEM images of the nanocarrier were made in order to validate the formulation of the NLC, to study the morphology, and to help to elucidate of the behavior of the formation. The technology did not require osmium contrast, as the analysis was performed using conventional protocols since the tucumã butter has diverse electron-dense components that contrast the image [13].

Figure 4

Figure (A,B) demonstrates the results of the IR spectrum. The NaDES and its constituents, betaine and malic acid (A), and the lipidic phase of the nanocarriers Jama and JamaNaDES are shown, along with NaDES spectrum profile (B). Diffractograms of the lipidic nanocarriers produced can be observed in (C): (a) Jama (absent of NaDES) and (b) JamaNaDES.

Figure 5

The cell viability as assessed by MTT assay after treatment with different concentrations of JamaNaDES for 24 h can be observed in (A). L132 cells were plated at a density of 1 × 10⁴ cells per well and treated with varying concentrations of JamaNaDES ranging from 214.87 mg/mL to 0.10 mg/mL. Following treatment, cells were incubated with MTT for 4 h, and absorbance was measured at 595 nm emission using a microplate reader. Cell viability values were calculated relative to the untreated control and expressed as a percentage. In (B), an overview graph of the Jama embryotoxicity test can be observed. In (C), the embryo hatching rates of zebrafish exposed to different concentrations of Jama for each observation time (hpf) are shown. Data are presented as mean ± standard error. Asterisks indicate significant differences between treatments and the control for a given time period (determined by Dunnett’s post-hoc comparison, p < 0.01 **; p < 0.001 ***). In (D), the mortality of zebrafish embryos exposed to different concentrations of Jama from 24 to 96 hpf is shown. Data are presented as mean ± standard error. Asterisks indicate significant differences between treatments and the control for a given time period (determined by Dunnett’s post-hoc comparison, p < 0.01 **; p < 0.001 ***). Dose-response curve (mortality) of organisms exposed to Jama for 96 h—LC50 44.9539 µg/mL—Model: sigmoidal—four parameters. R2 0.93 can be verified in (E). The graph of significant sublethal effects identified during the 96 h exposure to Jama can be found in (F). Data are presented as mean ± standard error. Asterisks indicate significant differenced between treatments and the control for a given time period (determined by Dunnett’s post-hoc comparison, p < 0.01 **; p < 0.001 ***). The rates of yolk sac edema (Yse) and pericardial edema (Pe) caused by Jama are shown in (G). Asterisks indicate significant differences between treatments and the control (determined by Dunnett’s post-hoc comparison, p < 0.05 *; p < 0.01 **; p < 0.001 ***). Error bars indicate standard error.

Figure 6

Photo documentation of organisms exposed for 96 h to concentrations of 0 mg/L (negative control), 20 µg/mL, 40 µg/mL, and 80 µg/mL of Jama can be observed in (A–D), respectively. Red arrows—a: darkening of the yolk sac; b: malabsorption syndrome; c: pericardial edema; d: yolk sac edema. Blue arrows—blood stasis. Green arrows—deformation in the notochord.

Figure 7

In (A), there is a graphic of the areas of the densitometry valleys for both the control group and the JamaNaDES treatment groups. The treatment group consists of the L132 cell supernatant treated with JamaNaDES at different concentrations. The asterisk indicates a significant difference between the groups (p < 0.05). In figure (B,C) the reactive oxygen species (ROS) production assessed using the CellROX^® Green Reagent assay after treatment with different concentrations of JamaNaDES for 24 h is shown. The data are presented as the raw values of GFP. In (B), there is a depiction of control cells and treated cells with ROS measurement, and in (C), there is the addition of H₂O₂ to observe action in a stressed environment. In (D), there are confocal fluorescence images of the experiment.

Figure 8

Morphological and physicochemical changes in Jama after different cooling methods. TEM micrographs of the three different cooling methods are observed in (A–C). In (D), the hydrodynamic diameter and polydispersity index are presented. In figure (A–C), the TEM microscopy of the nanoparticles obtained in protocols A, B, and C, respectively, are shown. Figure (D) displays the results obtained through the dynamic light scattering (DLS) technique, indicating the hydrodynamic diameters and polydispersity indices achieved using different cooling methodologies (A, B, and C).